The present invention relates to the inclusion of control reactions in real time reverse-transcriptase-initiated polymerase chains reactions, as applied to certain experiments in molecular biology.
The Polymerase Chain Reaction (PCR) is widely used to detect DNA sequences by amplifying their number by an exponential cyclic biochemical process. Purified RNA may be transcribed using enzymes known as reverse transcriptases to generate a DNA sequence that is complementary to all or part of the RNA. Such DNA made by reverse transcription, especially when the RNA that is reverse transcribed is mRNA, is known as complementary DNA (cDNA), which may then be amplified using PCR. This combined process is known as RT-PCR. As PCR proceeds, the number of copies of the amplified DNA increases and a number of methods and instruments that automate amplification and measurement of the products of amplification have been developed and are widely used. By continually monitoring in “real time” the progress of amplification reaction from its start, the number of DNA molecules present at the start of the process can be determined. This method is generally know as Quantitative Real-Time PCR and is an exceptionally sensitive method for detecting and quantitating small numbers of DNA molecules of a particular sequence. If reverse transcription is carried out on a sample of RNA to generate cDNA and the cDNA so generated is then amplified by Quantitative Real Time PCR, then the number of RNA molecules of a particular sequence may be determined. This process is commonly referred to as Quantitative Real-Time RT-PCR.
For this method to be most quantitative and reliable, the inclusion of artificial control reactions among the experimental reactions is desired. These controls primarily address potential causes of spurious or inaccurate results. First, impurities may be carried through purification or otherwise enter the samples being assayed which may inhibit the PCR process. Second, impurities may be carried through the purification of RNA or otherwise enter the samples which inhibit the reverse transcription reaction. Third, genomic DNA may have been carried through the purification process, which is designed to eliminate it, and may be amplified at the final PCR step producing amplified product that may falsely indicate the existence of more mRNA of a particular sequence than is actually present in the sample. In any set of Quantitative Real Time RT-PCR assays, redundant controls addressing all three of the above concerns may be desired. Having these controls configured conveniently along side experimental assays in a format compatible with automated processing and data collection is further desired. In addition, it may be desirable to include in a set of assays control reactions that detect mRNAs that would be expected to be present at roughly predictable levels. These mRNAs commonly code for proteins that are involved in basic cellular metabolism or are components of ubiquitous and necessary cellular structures such as ribosomes or the cytoskeleton. Genes coding for such proteins are often referred to as housekeeping genes.
Quantitative Real-Time RT-PCR is widely used to monitor the levels of multiple mRNAs in tissues or other collections of cells. Often the various mRNAs being monitored in a single experiment are related. In such an experiment one wishes to monitor the relative levels the various mRNAs but must meet the underlying assumption that all samples being compared are of similar quality. The process of preparation of mRNA from cells, reverse transcription and amplification, however careful a practitioner may be, may be less than optimal or may proceed with variable efficiency. To control for this possibility, it is advisable to include one or more control reactions to monitor reaction efficiency and quality of sample preparation. Moreover, detection of these mRNAs at unexpectedly low levels may alert the experimenter to suboptimal quality of the assayed samples or their preparation. Certain mRNAs are expressed in substantially all cells of a species, sperm being a notable exception to this generalization, at high to moderate levels and detection of one or several of these mRNAs may be used to monitor purification yield and serve as reference levels for expression of mRNAs that one wishes to measure in one's assay. Genes and their mRNA transcripts that code for protein involved in fundamental cellular metabolism or are components of cellular structures are examples and are commonly referred to as housekeeping genes.